Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog

Dominika Borek, Karolina Michalska, Krzysztof Brzezinski, Agnieszka Kisiel, Jan Podkowinski, David T. Bonthron, Daniel Krowarsch, Jacek Otlewski, Mariusz Jaskolski

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, L1A) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (L1A) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg·L-1 of culture, respectively. The enzymes are heat-stable up to 60°C and show both isoaspartyl di-peptidase and L-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.

Original languageEnglish (US)
Pages (from-to)3215-3226
Number of pages12
JournalEuropean Journal of Biochemistry
Volume271
Issue number15
DOIs
StatePublished - Aug 2004

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Aspartylglucosylaminase
Amidohydrolases
Lupinus
Asparaginase
Asparagine
Escherichia coli
Purification
Catalyst activity
Peptide Hydrolases
Enzymes
Proteins
Threonine
Kinetic parameters
Potassium
Hot Temperature
Genes

Keywords

  • Asparaginase
  • Aspartylglucosaminidase
  • Glutathione
  • Isoaspartyl peptidase
  • Ntn-hydrolase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog. / Borek, Dominika; Michalska, Karolina; Brzezinski, Krzysztof; Kisiel, Agnieszka; Podkowinski, Jan; Bonthron, David T.; Krowarsch, Daniel; Otlewski, Jacek; Jaskolski, Mariusz.

In: European Journal of Biochemistry, Vol. 271, No. 15, 08.2004, p. 3215-3226.

Research output: Contribution to journalArticle

Borek, D, Michalska, K, Brzezinski, K, Kisiel, A, Podkowinski, J, Bonthron, DT, Krowarsch, D, Otlewski, J & Jaskolski, M 2004, 'Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog', European Journal of Biochemistry, vol. 271, no. 15, pp. 3215-3226. https://doi.org/10.1111/j.1432-1033.2004.04254.x
Borek, Dominika ; Michalska, Karolina ; Brzezinski, Krzysztof ; Kisiel, Agnieszka ; Podkowinski, Jan ; Bonthron, David T. ; Krowarsch, Daniel ; Otlewski, Jacek ; Jaskolski, Mariusz. / Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog. In: European Journal of Biochemistry. 2004 ; Vol. 271, No. 15. pp. 3215-3226.
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